Genetic and environmental factors are thought to contribute to Parkinson's disease. Mutated alpha-synuclein can cause familial PD and normal alpha-synuclein is found in Lewy bodies. Recent studies have indicated that certain pesticides can increase aggregation of alpha-synuclein and dopamine itself appears to promote the stabilization of the toxic alpha-synuclein protofibril. We now have evidence that alpha-synuclein directly interacts with the vesicular monoamine transporter (VMAT2), a key regulator of cytosolic dopamine, and may alter its function. Furthermore, several pesticides have been shown to inhibit VMAT2 function. Therefore, the purpose of the proposal is to investigate how the genetic and environmental regulation of VMAT2 may predispose the dopamine system in injury. Hypothesis 1: alpha-Synuclein and pesticides can directly alter VMAT2 function. Aim 1: alpha-Synuclein alters VMAT2 function. Wild type and mutant forms of VMAT2 and (alpha-synuclein will be cotransfected into COS-7 cells and tested for VMAT2-mediated dopamine uptake. In addition striatal synaptic vesicles will be isolated from alphaz-synuclein transgenic mice and tested for VMAT2 function. Aim 2: Pesticides alter VMAT2 function. The effects of organochlorine insecticides and rotenone will be tested using the techniques in Aim 1. Hypothesis 2: Pesticides and VMAT2 regulate alpha-synuclein. Aim 3: Pesticides alter alpha-synuclein expression and increase vulnerability to dopaminergic damage, alpha-Synuclein expression in mice treated with pesticides and/or MPTP will be assessed by laser capture microdissection, real time PCR, immunocytochemistry, and western blotting. Aim 4: Alteration of VMAT2 expression regulates alpha-synuclein expression. To determine if VMAT2 expression alters alpha-synuclein expression and localization we will examine mutant mice with reduced levels of VMAT2. Completion of the above specific aims will clarify the interactions among key genetic and environmental factors thought to be involved in the development of idiopathic Parkinson's disease. An improved understanding at the root causes of PD should aid in the development of therapeutic strategies and regulatory policies aimed at preventing the disease.